Molecularly-imprinted-polymer coated conductive nanoparticles for cotinine detection, and associated devices and methods

ABSTRACT

A device for detecting cotinine includes (a) a film that includes a plurality of molecularly-imprinted-polymer (MIP) coated conductive nanoparticles having specific affinity for binding with cotinine, and (b) two electrodes in contact with the film for passing electrical current through the film to detect binding with cotinine as a change in electrical conductivity of the film. A MIP coated conductive nanoparticle for detecting cotinine includes (a) a conductive nanoparticle, (b) a silicon dioxide coating formed on the conductive nanoparticle and forming a first shell around the conductive nanoparticle, and (c) an MIP coating formed on the silicon dioxide coating and forming the second shell, wherein the MIP coating includes a polymer molecularly imprinted with cotinine to provide specific affinity for binding of cotinine to the MIP coated conductive nanoparticle such that the cotinine is detectable as a change in electrical conductivity of the MIP coated conductive nanoparticle.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority from U.S.Provisional Application Ser. No. 62/318,700 filed Apr. 5, 2016, which isincorporated herein by reference in its entirety.

BACKGROUND

Cotinine is the predominant metabolite of nicotine. While nicotine ispresent in a subject only for a couple of hours after exposure tocigarette smoke, cotinine is present in a subject for several days afterexposure to cigarette smoke. Thus, the level of cotinine in the blood,urine, or saliva of a subject is an indication of the subject's exposureto cigarette smoke over a period of several days leading up to the test.Cotinine testing is most commonly used in smoking cessation programs tomonitor compliance. However, cotinine testing is also used in othersituations such as in employee wellness programs and for monitoringrequired in cases of court-mandated no-smoking directives. The mostwidely used commercially available cotinine tests are based on urinetesting. In these tests, sufficient concentration of cotinine in a urinesample results in a color change of a test strip. A human operator oruser reads this color change by visual inspection. A positive testresult occurs only at concentrations associated with first-handcigarette smoking.

SUMMARY

In an embodiment, a molecularly-imprinted-polymer (MIP) coatedconductive nanoparticle for detecting cotinine includes (a) a conductivenanoparticle capable of conducting an electrical current, (b) a silicondioxide coating formed on the conductive nanoparticle and forming afirst shell around the conductive nanoparticle, and (c) an MIP coatingformed on the silicon dioxide coating and forming the second shell. TheMIP coating includes a polymer molecularly imprinted with cotinine toprovide specific affinity for binding of cotinine to the MIP coatedconductive nanoparticle such that the cotinine is detectable as a changein electrical conductivity of the MIP coated conductive nanoparticle.

In an embodiment, a device for detecting cotinine includes (a) a filmincluding a plurality of MIP coated conductive nanoparticles havingspecific affinity for binding with cotinine, and (b) two electrodes incontact with the film for passing electrical current through the film todetect binding with cotinine as a change in electrical conductivity ofthe film.

In an embodiment, a method for detecting cotinine in a liquid sampleincludes exposing a film to the liquid sample. The film includes MIPcoated conductive nanoparticles having specific affinity for bindingwith cotinine, and the film has electrical conductivity sensitive tobinding with cotinine. The method further includes measuring electricalconductivity of the film, and determining the presence of cotinine inthe liquid sample based upon the electrical conductivity.

In an embodiment, a method for manufacturing a device for detectingcotinine includes (a) making a solution including a polymer, a solvent,and cotinine, and (b) adding silicon dioxide coated conductivenanoparticles to the solution to form a shell, including the polymer andthe cotinine, around each of the silicon dioxide coated metalnanoparticles through binding of the polymer to the silicon dioxide, soas to form polymer-silicon dioxide coated conductive nanoparticles. Themethod further includes extracting the polymer-silicon dioxide coatedconductive nanoparticles from the solution, and removing the cotininefrom the polymer-silicon dioxide coated conductive nanoparticles to formmolecularly-imprinted-polymer (MIP) coated conductive nanoparticleshaving (i) specific affinity for binding with cotinine and (ii)electrical conductivity sensitive to said binding with cotinine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a device for detecting cotinine in a liquid sample,according to an embodiment.

FIG. 2 is a schematic view of a device for detecting cotinine in aliquid sample, according to an embodiment.

FIG. 3 is a schematic view of another device for detecting cotinine in aliquid sample, according to an embodiment.

FIG. 4 illustrates a molecularly-imprinted-polymer coated conductivenanoparticle, according to an embodiment.

FIG. 5 illustrates a molecularly-imprinted-polymer coated conductivenanoparticle with a binding layer that aids binding of the molecularlyimprinted polymer to the conductive core, according to an embodiment.

FIGS. 6A and 6B illustrate a sensing unit for detection of cotinine,which includes interdigitated electrodes, according to an embodiment.

FIG. 7 illustrates a packaged sensing unit for detection of cotinine,which includes interdigitated electrodes, according to an embodiment.

FIG. 8 illustrates a method for detecting cotinine in a liquid sample,according to an embodiment.

FIG. 9 illustrates a method for producing molecularly-imprinted-polymercoated conductive nanoparticles configured for cotinine detection,according to an embodiment.

FIG. 10 illustrates a method for manufacturing a cotinine detectiondevice, according to an embodiment.

FIG. 11 shows exemplary data obtained for an embodiment of the device ofFIG. 1 implementing an example of the sensing unit of FIGS. 6A and 6Bwith molecularly imprinted polyvinylpyrrolidone-co-poly(methylmethacrylate) coated silicon dioxide coated gold nanoparticles.

DETAILED DESCRIPTION OF THE EMBODIMENTS

FIG. 1 illustrates one exemplary device 100 for detecting cotinine in aliquid sample 172. Liquid sample 172 is, for example, a urine sample ofa subject 170. In FIG. 1 shows two instances 100(1) and 100(2) of device100, two corresponding instances 172(1) and 172(2) of liquid sample 172,and two corresponding instances 170(1) and 170(2) of subject 170. In theexemplary scenarios shown in FIG. 1, subject 170(1) is a first-handcigarette smoker smoking a cigarette 180, while subject 170(2) isexposed only to second-hand cigarette smoke 182. Device 100 includesmolecularly-imprinted-polymer (MIP) coated conductive nanoparticles 110to detect the presence of cotinine in liquid sample 172 when placed inor on device 100. MIP coated conductive nanoparticles 110 are discussedin further detail below in reference to FIGS. 4 and 5. MIP coatedconductive nanoparticles 110 of devices 100(1) and 100(2) are labeled110(1) and 110(2), respectively. MIP coated conductive nanoparticles 110have (a) specific affinity for binding with cotinine and (b) electricalconductivity sensitive to the amount of cotinine bound thereto. Ameasurement of the electrical conductivity of MIP coated conductivenanoparticles 110 produces a test result 190. FIG. 1 shows two instances190(1) and 190(2) of test results 190, respectively produced by devices100(1) and 100(2).

Test result 190 indicates the presence of a detectable amount ofcotinine in liquid sample 172. Test results 190 may further indicate thedetected concentration of cotinine in liquid sample 172. Readout ofdevice 100 is performed electronically and does not require a subjectiveevaluation of device 100. This is in contrast to the conventionalcolor-change based cotinine tests available on the commercial market,which suffer from human subjectivity in the visual readout process.Device 100 eliminates the subjectivity of a human readout and is atleast for this reason more reliable than the conventional tests.

In addition, at least in part by virtue of MIP coated conductivenanoparticles 110, device 100 is more sensitive than the conventionaltests and is capable of detecting cotinine in liquid sample 172 at lowconcentrations associated only with second-hand smoking, such asexperienced by subject 170(2). Furthermore, in certain embodiments, testresult 190 indicates the concentration of cotinine in liquid sample 172,either as a relative measure or an absolute measure, such that device100 is capable of distinguishing between first-hand smoking, such as bysubject 170(1), and second-hand smoking, such as by subject 170(2). Suchembodiments of device 100 may further be capable of indicating theamount of first-hand smoking and/or the severity of second-hand smoking.In an embodiment, device 100 is calibrated such that test result 190indicates the cotinine concentration of liquid sample 172 in absoluteterms, for example in units of nanograms per milliliter (ng/ml).Alternatively, test result 190 may indicate the cotinine concentrationof liquid sample 172 in relative terms.

In one embodiment, device 100 is configured for single-use and isdiscarded after testing of a single liquid sample 172. In anotherembodiment, device 100 is configured for repeated use. In thisembodiment, device 100 is washed after testing of each liquid sample 172to remove cotinine bound to MIP coated conductive nanoparticles 110 ofdevice 100 for unbiased testing of the next liquid sample 172.

FIG. 2 is a schematic view of one exemplary device 200 for detectingcotinine in liquid sample 172. Device 200 is an embodiment of device100, and may implement any one of the embodiments of device 100discussed above in reference to FIG. 1. Device 200 includes a film 210containing MIP coated conductive nanoparticles 110. Device 200 furtherincludes two electrodes 220 in contact with film 210, and conductivitymeasurement circuitry 240 communicatively coupled with each electrode220. By virtue of MIP coated conductive nanoparticles 110, film 210 has(a) specific affinity for binding with cotinine and (b) electricalconductivity sensitive to the amount of cotinine bound thereto.

In operation, liquid sample 172 is placed in device 200 in contact withfilm 210 such that cotinine in liquid sample 172 may bind to MIP coatedconductive nanoparticles. Conductivity measurement circuitry 240measures the conductivity of film 210 between electrodes 220 to detectcotinine from liquid sample 172 bound to film 210. Conductivitymeasurement circuitry 240 includes an interface 232. Interface 232outputs test result 190. Test result 190 includes a measurement made byconductivity measurement circuitry 240, and/or one or more parametersderived from such measurement.

Herein, measurement of conductivity may refer to measurement of actualconductivity or measurement of a parameter related to conductivity, suchas resistivity, resistance, and conductance. In an embodiment,conductivity measurement circuitry 240 is configured to pass a currentthrough film 210 between electrodes 220 to measure the resistance offilm 210 between electrodes 220. In one embodiment, test result 190includes the conductivity or resistivity of film 210. In anotherembodiment, test result 190 includes the conductance or resistance offilm 210 between electrodes 220. In yet another embodiment, test result190 includes another parameter derived from the conductivity,resistivity, conductance, or resistance of film 210, such as aconcentration of cotinine in liquid sample 172.

In certain implementations, conductivity measurement circuitry 240 isconfigured to pass a direct current (DC) through film 210 betweenelectrodes 220 to measure the DC resistance or DC conductance of film210 between electrodes 220.

In certain embodiments, device 200 includes a substrate 230 thatsupports electrodes 220 and film 210. Although shown in FIG. 2 as beingplanar, film 210 may deviate from being planar, without departing fromthe scope hereof. For example, in embodiments with electrodes 220disposed between substrate 230 and film 210, film 210 may occupy atleast some of gap 222. Furthermore, film 210 need not fully coverelectrodes 220. Film 210 may have thickness 212 in the range from 100nanometers to 1 micron. In one example, thickness 212 is in the rangefrom 400 to 600 nanometers.

In one embodiment, film 210 may be composed of MIP coated conductivenanoparticles 110. In another embodiment, film 210 includes MIP coatedconductive nanoparticles 110. For example, film 210 may be mostlycomposed of MIP coated conductive nanoparticles 110 but also includesome polymers not bound to MIP coated conductive nanoparticles 110.

Optionally, device 200 includes an enclosure 280 that houses film 210,electrodes 220, conductivity measurement circuitry 240, and substrate230 (if included). Enclosure 280 forms a receptacle 290 configured toreceive liquid sample 172 to hold liquid sample 172 in contact with film210. The volume capacity of receptacle 290 is for example in the rangefrom 0.5 to 5 milliliters. This volume is sufficient for device 200 toreliably detect cotinine in liquid sample 172.

FIG. 3 is a schematic view of another exemplary device 300 for detectingcotinine in a liquid sample 172. Device 300 is an embodiment of device100, and may implement any one of the embodiments of device 100discussed above in reference to FIG. 1. Device 300 is similar to device200 except that device 300 need not include conductivity measurementcircuitry 240. Embodiments of device 300 that do not includeconductivity measurement circuitry 240 may be configured to cooperatewith third party conductivity measurement circuitry 240 to produce testresult 190. Such third party conductivity measurement circuitry 240 maybe placed externally to enclosure 280.

FIG. 4 illustrates one exemplary MIP coated conductive nanoparticle 400which is an embodiment of MIP coated conductive nanoparticle 110 and maybe implemented in film 210. MIP coated conductive nanoparticle 400includes a conductive core 410 and a MIP shell 420 around core 410. MIPshell 420 is a coating that is composed of, or includes, molecularlyimprinted polymers, imprinted with cotinine, such that MIP shell 420 hasspecific affinity for binding with cotinine. Conductive core 410 iselectrically conductive. However, the conductivity of MIP coatedconductive nanoparticle 400 is affected by MIP shell 420. Theresistivity of MIP shell 420 increases with the amount of cotinine boundthereto, such that the conductivity of MIP coated conductivenanoparticle 400 decreases with the amount of cotinine bound thereto.Thus, when MIP coated conductive nanoparticles 400 are implemented indevice 100, for example in film 210 of device 200 or 300, theconductivity of film 210 decreases with the amount of cotinine in liquidsample 172.

In one embodiment, conductive core 410 is a metal core essentiallyconsisting of a metal such as gold, silver, platinum, or a combinationthereof. In another embodiment, conductive core 410 includes both metal(such as gold, silver, platinum, or a combination thereof) andnon-conductive material. In yet another embodiment, conductive core 410includes a non-metallic conductive material such as graphite, optionallyin combination with conductive metal and/or non-conductive material.

MIP shell 420 includes a polymer that is capable of hydrogen bondingwith cotinine to facilitate affinity for binding of cotinine to MIPshell 420. In one embodiment, MIP shell 420 includes, or issubstantially composed of, a homopolymer molecularly imprinted withcotinine and capable of hydrogen bonding. For example, MIP shell 420 maybe substantially composed of polyvinylpyrrolidone (PVP) or poly(methylmethacrylate) (PMMA), each molecularly imprinted with cotinine. Inanother embodiment, MIP shell 420 includes, or is substantially composedof a copolymer molecularly imprinted with cotinine and capable ofhydrogen bonding. As compared to homopolymer based embodiments of MIPshell 420, the copolymer may enhance the sensitivity of MIP shell 420 tobinding of cotinine therewith. Thus, copolymer based embodiments of MIPshell 420 may exhibit increased efficiency for binding with cotinine,and hence increased sensitivity, such that devices 100 utilizing acopolymer based embodiment of MIP shell 420 may be capable of detectinglower cotinine concentrations, as compared to devices 100 utilizing ahomopolymer based embodiment of MIP shell 420. In one implementation,MIP shell 420 is substantially composed of PVP-co-PMMA.

Core 410 may have diameter 412 in the range from 10 to 100 nanometers.MIP shell 420 may have thickness 422 in the range from 10 to 50nanometers.

FIG. 5 illustrates one exemplary MIP coated conductive nanoparticle 500with a binding layer that aids binding of the molecularly imprintedpolymer to the conductive core. MIP coated conductive nanoparticle 500is an embodiment of MIP coated conductive nanoparticle 110, which issimilar to MIP coated conductive nanoparticle 400. MIP coated conductivenanoparticle 500 includes conductive core 410, MIP shell 420, and abinding layer 510 disposed on conductive core 410 between conductivecore 410 and MIP shell 420. Binding layer 510 forms an inner shellaround conductive core 410. Binding layer 510 aids the binding of thepolymer of MIP shell 420 to conductive core 410.

In one embodiment, binding layer 510 is substantially composed ofsilicon dioxide (i.e., silica). In another embodiment, binding layer 510is substantially composed of alumina. In yet another embodiment, bindinglayer 510 includes silicon dioxide and/or alumina. Binding layer 510 mayhave thickness 512 of about 10 nanometers, for example in the range from3 to 30 nanometers.

FIGS. 6A and 6B illustrate one exemplary sensing unit 600 withinterdigitated electrodes. Sensing unit 600 may be implemented in device100. Sensing unit 600 forms an embodiment of film 210, electrodes 220,and substrate 230 of devices 200 and 300. FIGS. 6A and 6B shows sensingunit 600 in orthogonal cross-sectional views. FIG. 6A is a view of across section taken along line 6A-6A′ in FIG. 6B. FIG. 6B is a view of across section taken along line 6B-6B′ in FIG. 6A. FIGS. 6A and 6B arebest viewed together.

Sensing unit 600 includes a substrate 630, interdigitated electrodes620, and film 210. Substrate 630 is an embodiment of substrate 230.Interdigitated electrodes 620 are an embodiment of electrodes 220. Eachelectrode 620 has a plurality of fingers 624. For clarity ofillustration, not all fingers 624 are labeled in FIGS. 6A and 6B.Fingers 624 of one electrode 620 are interdigitated with fingers 624 ofthe other electrode 620. Since electrodes 620 are interdigitated,sensing unit 600 facilitates a high degree of spatial averaging over theextent of film 210, in a manner that is conceptually equivalent tomaking many parallel conductivity measurements for different portions offilm 210. This helps ensure high accuracy of a conductivity measurementmade by, for example conductivity measurement circuitry 240.

Each finger 624 has height 622, width 626, and length 627. Height 622 isfor example in the range from 50 to 200 nanometers, such asapproximately 100 nanometers. Width 626 is for example in the range from10 to 100 microns, such as approximately 40 microns. Length 627 is forexample in the range from 0.5 to 3 centimeters, such as approximately 2centimeters. Adjacent fingers 624 are spaced apart from each other by aspacing 628. Spacing 628 is for example in the range from 5 to 50microns, such as approximately 20 microns. Each electrode 620 mayinclude between 50 and 300 fingers 624, for example approximately 150fingers 624.

Substrate 630 is a dielectric material such as silicon dioxide.Interdigitated electrodes 620 are for example metallic. In oneembodiment, interdigitated electrodes 620 are deposited on substrate 630using photolithography. Interdigitated electrodes 620 may be chromiumelectrodes, optionally with a nickel overlayer between the chromium andfilm 210. Substrate 630 has thickness 612 sufficient to avoid breakageof sensing unit 600. Thickness 612 may be in the range from 0.75 to 1.0millimeters.

The transverse extents 614 (only one shown in FIG. 6B) are configured toprovide a contact area between liquid sample 172 and film 210sufficiently large to (a) accommodate a sample volume sufficient fordetection of cotinine at a desired sensitivity level while (b) keepingthe diffusion time for cotinine in liquid sample 172 to the surface offilm 210 sufficiently low to detect cotinine in liquid sample, andoptionally measure its concentration, within a desired duration. In oneexample, the surface area of film 210 is in the range between 200 and1000 millimeters². With a surface area of 400 millimeters², film 210 ofsensing unit 600 may accommodate a 1.0 milliliter liquid sample 172 witha column height of 2.5 millimeters.

Without departing from the scope hereof, transverse extent 614 ofsubstrate 630 may exceed transverse extent 614 of interdigitatedelectrodes 620 and/or transverse extent 614 of interdigitated electrodes620 may exceed transverse extent 614 of film 210.

FIG. 7 illustrates one exemplary packaged sensing unit 700. Sensing unit700 is an extension of sensing unit 600, which further includes anenclosure 780. Sensing unit 700 may be implemented in device 100.Sensing unit 700 forms an embodiment of film 210, electrodes 220,substrate 230, and enclosure 280 of devices 200 and 300. Enclosure 780contains sensing unit 600. Optionally, sensing unit 700 further includesconductivity measurement circuitry 240 communicatively withinterdigitated electrodes 620 and contained within enclosure 780.Enclosure 780 is configured with an opening 790 over film 210. Opening790 has height 782 and forms a receptacle for receiving liquid sample172 and holding liquid sample 172 in contact with film 210. Thisreceptacle is an embodiment of receptacle 290.

In one example, height 782 is at least 1 millimeter. In another example,height 782 is at least 2.5 millimeters. In yet another example, height782 is at least 5 milliliters.

FIG. 8 illustrates one exemplary method 800 for detecting cotinine inliquid sample 172. Method 800 may be performed by device 100, or one ofdevices 200 and 300, optionally implementing either one of sensing units600 and 700.

In a step 810, a film that includes MIP coated conductive nanoparticles110 is exposed to liquid sample 172. Step 810 may include a step 812 ofreceiving liquid sample 172. In one example of step 810, liquid sample172 is placed in receptacle 290 of device 200 or 300 and in contact withfilm 210.

A step 820 measures the electrical conductivity of the film of step 810.In one example of step 820, conductivity measurement circuitry 240measures the conductivity of film 210 between electrodes 220. Step 820may include a step 822 of passing current through the film. Step 822 isfor example performed by conductivity measurement circuitry 240 viaelectrodes 220. In certain embodiments, step 822 includes a step 824 ofpassing electrical current through the film between interdigitatedelectrodes. In one example of step 824, conductivity measurementcircuitry 240 passes current through film 210 between interdigitatedelectrodes 620. Optionally, step 820 implements a step 826 of measuringthe conductivity of the film in the form of resistance. In one exampleof step 826, conductivity measurement circuitry 240 measures theresistance of film 210 between electrodes 220 or between interdigitatedelectrodes 620.

In a step 830, method 800 determines the presence (or absence) ofcotinine in liquid sample 172 based upon the conductivity measured instep 820. In one example of step 830, conductivity measurement circuitry240 outputs a test result 190 based upon the conductivity measured instep 820, wherein the test result 190 indicates if cotinine was or wasnot present (at a detectable level) in liquid sample 172. In certainembodiments, step 830 includes a step 832 of deriving the cotinineconcentration in liquid sample 172 from the measured conductivity. Inone example of step 830, conductivity measurement circuitry 240 derivesthe cotinine concentration in liquid sample 172 from the conductivitymeasured in step 820. In this example, conductivity measurementcircuitry 240 may output the cotinine concentration as test result 190.In another example of step 830, a user derives the cotinineconcentration in liquid sample 172 from test result 190 produced bydevice 100. The user may utilize a lookup table supplied to the usertogether with device 100 to convert test result 190 to a cotinineconcentration.

FIG. 9 illustrates one exemplary method 900 for producing MIP coatedconductive nanoparticles configured for cotinine detection, such as MIPcoated conductive nanoparticles 110 or 500. Method 900 is for exampleused to manufacture device 100.

A step 910 forms a solution including polymer, a solvent, and cotinine.Step 910 dissolves the polymer and cotinine in the solvent andoptionally mixes the solution for a period of time. In one example ofstep 910, the polymer is dissolved in dichloromethane anddimethylformamide with cotinine. In certain embodiments, step 910implements a step 912 of including a polymer capable of hydrogen bondingwith cotinine. Examples of such polymers include PVP and PMMA, andcombinations thereof. Step 910 may implement a step 914 of including acopolymer to enhance the sensitivity of the MIP coating, of the MIPconductive nanoparticles produced by method 900, to binding of cotininetherewith. In one embodiment, step 910 implements both step 912 and step914. In an example of this embodiment, PVP-co-PMMA and cotinine aredissolved in a solvent, and optionally mixed for a period of time. Asdiscussed above in reference to FIG. 4, PVP-co-PMMA is capable ofhydrogen bonding with cotinine. PVP-co-PMMA also demonstrates enhancedsensitivity to binding of cotinine therewith as compared to PVP alone orPMMA alone.

In a step 920, method 900 adds silicon dioxide coated conductivenanoparticles to the solution formed in step 910. Each silicon dioxidecoated nanoparticles is, for example, core 410 with binding layer 510,wherein binding layer 510 is substantially composed of silicon dioxide.Optionally, a catalyst is added to the solution to facilitate binding ofthe polymer to the silicon dioxide. While in the solution, the silicondioxide coated conductive nanoparticles are coated with the polymer andcotinine through binding of the polymer to the silicon dioxide. Step 920thus forms a shell around each silicon dioxide coated nanoparticle. Theproduct of step 920 is a solution that contains polymer-silicon dioxidecoated conductive nanoparticles. For each polymer-silicon dioxide coatedconductive nanoparticle, the polymer forms an outer shell around thesilicon dioxide shell, and the polymer shell has cotinine embeddedtherein. The silicon dioxide-coated conductive nanoparticles are, forexample, silicon dioxide-coated metal nanoparticles, wherein each metalnanoparticle includes or is composed of gold, silver, and or platinum,for example. Alternatively, the silicon dioxide-coated conductivenanoparticles are non-metallic conductive nanoparticles (such asdiscussed above in reference to FIG. 4) coated with silicon dioxide.

In certain embodiments, method 900 includes a step 916 that is performedbetween steps 910 and 920. In step 916, method 900 mixes a cross-linkingagent into the solution to aid binding of the polymer to the silicondioxide coated conductive nanoparticles in step 920. The cross-linkingagent is for example tetraethyl orthosilicate (TEOS). The cross-linkingagent may cooperate with the optional catalyst of step 920 to aidbinding of the polymer to the silicon dioxide in step 920.

In a step 930, method 900 extracts the polymer-silicon dioxide coatedconductive nanoparticles from the solution. Step 930 may implement astep 932 of centrifuging the solution to separate out thepolymer-silicon dioxide coated conductive nanoparticles.

A step 940 removes the cotinine from the polymer-silicon dioxide coatedconductive nanoparticles, of step 930, to form MIP coated conductivenanoparticles having (a) specific affinity for binding with cotinine and(b) electrical conductivity sensitive to the amount of cotinine boundthereto. Each MIP coated conductive nanoparticles thus formed has aconductive core surrounded by an inner silicon dioxide shell that issurrounded by an outer MIP shell, molecularly imprinted with cotinine.In one example, step 940 forms MIP coated conductive nanoparticle 500with binding layer 510 being substantially composed of silicon dioxide.

In one embodiment, step 940 includes steps 942 and 944. In step 942, thepolymer-silicon dioxide coated conductive nanoparticles extracted instep 930 are washed in a solvent to remove the cotinine from the polymershell of each polymer-silicon dioxide coated conductive nanoparticle toform the MIP coated conductive nanoparticles. In one example, thesolvent used in step 942 is an aromatic solvent such as toluene, xylene,benzene, or a combination thereof. In step 944, this solution iscentrifuged to separate out the MIP coated conductive nanoparticles.

Optionally, method 900 includes a step 950 of collecting and drying theMIP coated conductive nanoparticles. In one example, step 950 dries theMIP coated conductive nanoparticles in vacuum.

In an alternative embodiment, method 900 utilizes alumina coatedconductive nanoparticles instead of silicon dioxide coated conductivenanoparticles.

In one example of method 900, 0.5 grams of PVP-co-PMMA and 0.25 grams ofcotinine are dissolved in 3 milliliters of dichloromethane and 2milliliters dimethylformamide in step 910. Also as part of step 910,this solution is stirred continuously for several hours, such as atleast 6 hours. Next, in step 916 of this example of method 900, 1milliliter of TEOS is added to the solution. This addition may beperformed as a dropwise addition over 5 minutes. Subsequently, in step920 of this example of method 900, 0.01 grams of silicon dioxide coatedgold nanoparticles (AuNP@SiO₂-NP) and 0.2 milliliters of 0.01 molehydrochloric acid are added to the solution, and the solution is stirredfor several hours, such as at least 6 hours. Hydrochloric acid functionsas a catalyst for the TEOS polymerization in step 920. In this example,step 920 forms PVP-co-PMMA coated silicon dioxide coated goldnanoparticles, wherein the PVP-co-PMMA layer has cotinine embeddedtherein. Step 930 of this example of method 900 implements an example ofstep 932, wherein the solution is centrifuged at 5000 revolutions perminute for 5 minutes to separate out the PVP-co-PMMA coated silicondioxide coated gold nanoparticles. Step 940 of this example of method900 implements steps 942 and 944. In this example, the PVP-co-PMMAcoated silicon dioxide coated gold nanoparticles are washed with toluenein step 942 to remove the cotinine, to form molecularly imprintedPVP-co-PMMA coated silicon dioxide coated gold nanoparticles. Step 944then separates out the molecularly imprinted PVP-co-PMMA coated silicondioxide coated gold nanoparticles from the solution by centrifugation instep 944. Each of these molecularly imprinted PVP-co-PMMA coated silicondioxide coated gold nanoparticles are an embodiment of MIP coatedconductive nanoparticle 500 with core 410 being gold, binding layer 510being silicon dioxide, and MIP shell 420 being PVP-co-PMMA molecularlyimprinted with cotinine. Optionally, the removal of cotinine isconfirmed with ultraviolet-visible spectroscopy after step 942. In step950 of this example of method 900, the molecularly imprinted PVP-co-PMMAcoated silicon dioxide coated gold nanoparticles are collected and driedin vacuum.

Although not shown in FIG. 9, method 900 may include producing thesilicon dioxide coated conductive nanoparticles prior to performing step910. In one example, citrate-stabilized gold nanoparticles are producedby the method described in the literature (see, for example, Frens, G.“Controlled nucleation for regulation of particle-size in monodispersegold suspensions”, Nat.-Phys. Sci. (1973) 241, 20-22). A 0.5 millimoleaqueous solution of HAuCl₄.3H₂O (Sigma-Aldrich) is boiled for 20 minuteswith 1.5 millimole Na₃C₆H₅O₇ (Sigma-Aldrich). During this heatingperiod, the solution color becomes wine red indicating the presence ofthe nanoparticles. The solution of colloidal gold is allowed to cool toroom temperature. The sol-gel process (Kobayashi, et al., “Sol-gelprocessing of silica-coated gold nanoparticles”, Langmuir (2001) 176375-79) may be used to synthesize the AuNP@SiO₂ core-shellnanoparticles. A sample of 0.1 grams of the gold nanoparticles aresuspended in 20 milliliters of a 4:1 mixture of ethanol and deionizedwater and sonicated for 30 minutes. At the end of the sonication period,1 milliliter of a 25% aqueous ammonia solution and 0.8 milliliters ofTEOS are added and the mixture is allowed to react for 12 hours withstirring at room temperature. The resulting silicon dioxide coated goldnanoparticles may be collected by centrifugation at 5000 rpm for 10minutes, rinsed with deionized water, and dried using vacuum.

FIG. 10 illustrates one exemplary method 1000 for manufacturing acotinine detection device. Method 1000 may for example be used tomanufacture device 100.

In a step 1030, method 1000 deposits MIP coated conductive nanoparticleson a substrate to form a film that includes the MIP coated conductivenanoparticles. In one example, step 1030 deposits MIP coated conductivenanoparticles 110 on substrate 230 to form film 210. In certainembodiments, step 1030 includes steps 1032 and 1034. Step 1032 dispersesthe MIP coated conductive nanoparticles in a solvent to form a MIPcoated conductive nanoparticle solution. In one example of step 1032,MIP coated conductive nanoparticles 110 are dispersed in a solvent, suchas dimethylformamide For example, the molecularly imprinted PVP-co-PMMAcoated silicon dioxide coated gold nanoparticles discussed above inreference to FIG. 9, and produced in the example based upon 0.5 grams ofPVP-co-PMMA, may be dispersed in 5 milliliters of dimethylformamide Step1034 deposits the solution, formed in step 1032, on the substrate.Optionally, step 1034 includes a step 1036 of drop casting the solutiononto the substrate. In one example of step 1034, a 100 microliteraliquot of dispersed molecularly imprinted PVP-co-PMMA coated silicondioxide coated gold nanoparticles (dispersed in 5 milliliters ofdimethylformamide) is drop cast onto substrate 630 over interdigitatedelectrodes 620, wherein substrate 630 and interdigitated electrodes 620have been pretreated with formic acid, deionized water and acetoneallowed to air dry prior to drop casting.

Step 1030 produces an embodiment of at least a portion of device 100,such as (a) a sensing unit including substrate 230, electrodes 220, andfilm 210 forming at least a portion of device 200 or of device 300, (b)sensing unit 600, or (c) sensing unit 700.

Step 1030 may be preceded by a step 1010 of synthesizing the MIP coatedconductive nanoparticles. In one embodiment, step 1010 is or includesmethod 900. Step 1010 produces MIP coated conductive nanoparticles 110,for example

Step 1030 may also be preceded by a step 1020 of forming electrodes onthe substrate. In one example, step 1020 forms electrodes 220 onsubstrate 230. In certain embodiments, step 1020 implements a step 1022of forming the electrodes as interdigitated electrodes. In one example,step 1022 forms interdigitated electrodes 620 on substrate 630.

In embodiment of method 1000 that include step 1020, step 1030implements a step 1038 of depositing the MIP coated conductivenanoparticles on the substrate over the electrodes. In one example ofstep 1038, MIP coated conductive nanoparticles 110 are deposited onsubstrate 230, including over electrodes 220, to form film 210, asdiscussed above in reference to FIG. 2. In another example of step 1038,MIP coated conductive nanoparticles 110 are deposited on substrate 630,including over interdigitated electrodes 620, to form film 210, asdiscussed above in reference to FIGS. 6A and 6B.

In an embodiment, method 1000 further includes a step 1040 of packagingthe substrate, electrodes, and film, and optionally conductivitymeasurement circuitry to form a packaged device for detecting cotinine.In one example of step 1040, the sensing unit formed in step 1030 ispackaged in enclosure 280, optionally together with conductivitymeasurement circuitry 240.

FIG. 11 shows exemplary data obtained for an embodiment of device 100implementing an example of sensing unit 600 with molecularly imprintedPVP-co-PMMA coated silicon dioxide coated gold nanoparticles. Themolecularly imprinted PVP-co-PMMA coated silicon dioxide coated goldnanoparticles are produced as discussed above in reference to FIG. 9 andincorporated in a cotinine detection device manufactured according tomethod 1000, as discussed above in reference to FIG. 10. The data ofFIG. 11 was obtained using a multimeter (Keithley model 2000) as anembodiment of conductivity measurement circuitry 240.

The device associated with the data of FIG. 11 had interdigitatedelectrodes 620, and each electrode 620 had 158 fingers 624. Height 622,width 626, and spacing 628 were 100 nanometers, 40 microns, and 20microns, respectively. Film 210 had thickness 212 of about 500nanometers.

The multimeter measured the resistance between interdigitated electrodes620 as a function of cotinine concentration in a liquid sample 172deposited onto the film. FIG. 11 plots the relative resistance anddemonstrates that this example of device 100 is sensitive to cotinine atconcentrations at least as low as 5 parts per billion (ppb). (Herein,“relative resistance” refers to (R_(c)−R₀)/R₀, wherein R_(c) is theresistance measured at the concentration indicated on the horizontalaxis and R₀ is the resistance measured before exposing the device tothis cotinine concentration.) This sensitivity is sufficient to detectsecond-hand smoking, which generally results in a cotinine concentrationin urine in the range between 10 and 30 nanograms/milliliter (10 to 30ppb). Furthermore, FIG. 11 shows that the device is capable ofquantitatively and accurately distinguish different concentrations atleast in the range from 5 ppb to 20 ppb. In this range, the devicedisplays a nearly linear relationship between the measured resistanceand the cotinine concentration. Additional measurements made in therange from 20 ppb to 200 ppb, not shown in FIG. 11, shows that therelative resistance quantitatively depends on the cotinine concentrationalso in this range.

Combinations of Features

Features described above as well as those claimed below may be combinedin various ways without departing from the scope hereof. For example, itwill be appreciated that aspects of one MIP coated conductivenanoparticle for cotinine detection, or associated method or device,described herein, may incorporate or swap features of another MIP coatedconductive nanoparticle for cotinine detection, or associated method ordevice, described herein. The following examples illustrate somepossible, non-limiting combinations of embodiments described above. Itshould be clear that many other changes and modifications may be made tothe systems and methods herein without departing from the spirit andscope of this invention:

(A1) A MIP coated conductive nanoparticle for detecting cotinine mayinclude (a) a conductive nanoparticle capable of conducting anelectrical current, (b) a silicon dioxide coating formed on theconductive nanoparticle and forming a first shell around the conductivenanoparticle, and (c) an MIP coating formed on the silicon dioxidecoating and forming the second shell, wherein the MIP coating includes apolymer molecularly imprinted with cotinine to provide specific affinityfor binding of cotinine to the MIP coated conductive nanoparticle suchthat the cotinine is detectable as a change in electrical conductivityof the MIP coated conductive nanoparticle.

(A2) In the MIP coated conductive nanoparticle denoted as (A1), theconductive nanoparticle may be a metal nanoparticle.

(A3) In the MIP coated conductive nanoparticle denoted as (A2), themetal nanoparticle may include gold.

(A4) In any of the MIP coated conductive nanoparticles denoted as (A1)through (A3), the polymer may be a dielectric polymer.

(A5) In the MIP coated conductive nanoparticle denoted as (A4), thedielectric polymer may be capable of hydrogen bonding with the cotinine.

(A6) In either or both of the MIP coated conductive nanoparticlesdenoted as (A4) and (A5), the dielectric polymer may include acopolymer.

(A7) In the MIP coated conductive nanoparticle denoted as (A6), thecopolymer may be polyvinylpyrrolidone-co-poly(methyl methacrylate).

(A8) In either or both of the MIP coated conductive nanoparticlesdenoted as (A6) and (A7), the copolymer may be capable of hydrogenbonding with the cotinine.

(B1) A device for detecting cotinine may include a film including aplurality of MIP coated conductive nanoparticles having specificaffinity for binding with cotinine, and two electrodes in contact withthe film for passing electrical current through the film to detect saidbinding as a change in electrical conductivity of the film.

(B2) In the device denoted as (B1), each of the MIP coated conductivenanoparticles may include (a) a conductive nanoparticle for providingelectrical conductivity to the MIP coated conductive nanoparticle, and(b) an MIP coating forming an outer shell around the conductivenanoparticle, the MIP coating including a polymer molecularly imprintedwith cotinine to provide specific affinity for binding of cotinine tothe MIP coated conductive nanoparticle.

(B3) In the device denoted as (B2), each of the MIP coated conductivenanoparticles may further include a silicon dioxide coating disposed onthe conductive nanoparticle and forming an intermediate shell around theconductive nanoparticle between the conductive nanoparticle and theouter shell, to enable formation of the MIP coating.

(B4) In either or both of the devices denoted as (B2) and (B3), theconductive nanoparticle may be a metal nanoparticle.

(B5) In the device denoted as (B4), the metal nanoparticle may includegold.

(B6) In any of the devices denoted as (B1) through (B5), the polymer maybe a dielectric polymer.

(B7) In the device denoted as (B6), the dielectric polymer may becapable of hydrogen bonding with the cotinine.

(B8) In either or both of the devices denoted as (B6) and (B7), thedielectric polymer may include a copolymer.

(B9) In the device denoted as (B8), the copolymer may bepolyvinylpyrrolidone-co-poly(methyl methacrylate).

(B10) In either or both of the devices denoted as (B8) and (B9), thecopolymer may be capable of hydrogen bonding with the cotinine.

(B11) In any of the devices denoted as (B1) through (B10), theelectrodes may be interdigitated electrodes.

(B12) In the device denoted as (B11), each of the interdigitatedelectrodes may have finger width in the range from 10 to 100 microns,and the spacing between the interdigitated electrodes may be in therange from 5 to 50 microns.

(B13) In any of the devices denoted as (B1) through (B12), the film mayhave thickness in the range from 100 nanometers to 1 micron.

(B14) Any of the devices denoted as (B1) through (B13) may furtherinclude a receptacle for receiving a liquid sample and placing theliquid sample in contact with the film to determine presence of cotininein the liquid sample.

(B15) The device denoted as (B14) may further include an enclosure forcontaining the film and the interdigitated electrodes, wherein thereceptacle is implemented as an opening formed in the enclosure abovethe film.

(B16) In either or both of the devices denoted as (B14) and (B15), thereceptacle may have volume capacity in the range between 0.5 and 5milliliters.

(B17) In any of the devices denoted as (B1) through (B16), the film andthe interdigitated electrodes may be cooperatively configured to producea linear relationship between (i) resistance measured between theinterdigitated electrodes and (ii) concentration of cotinine in a liquidsample placed in contact with the film.

(B18) In any of the devices denoted as (B1) through (B17), the MIPcoated conductive nanoparticles may include any one of the MIP coatedconductive nanoparticles denoted as (A1) through (A8).

(C1) A method for detecting cotinine in a liquid sample may include (a)exposing a film to the liquid sample, wherein the film includes MIPcoated conductive nanoparticles having specific affinity for bindingwith cotinine, the film having electrical conductivity sensitive to saidbinding, (b) measuring electrical conductivity of the film, and (c)determining presence of cotinine in the liquid sample based upon theelectrical conductivity.

(C2) In the method denoted as (C1), the step of measuring may includepassing electrical current through the film between two interdigitatedelectrodes in contact with the film.

(C3) In either or both of the methods denoted as (C1) and (C2), the stepof measuring may include measuring the electrical conductivity in formof resistance of the film between the interdigitated electrodes.

(C4) In the method denoted as (C3), the step of determining may furtherinclude deriving concentration of cotinine in the liquid sample from theresistance measured in the step of measuring.

(C5) In any of the methods denoted as (C1) through (C4), the step ofexposing may further include receiving the liquid sample.

(C6) In the method denoted as (C5), the step of receiving may includereceiving a volume of the liquid sample in range from 0.5 to 5milliliters.

(C7) In any of the methods denoted as (C1) through (C6), each MIP coatedconductive nanoparticle may include (i) a conductive nanoparticle forproviding electrical conductivity to the MIP coated conductivenanoparticle, and (ii) an MIP coating forming an outer shell around theconductive nanoparticle, the MIP coating including a polymer molecularlyimprinted with cotinine to provide specific affinity for binding ofcotinine to the MIP coated conductive nanoparticle.

(C8) In the method denoted as (C7), each of the MIP coated conductivenanoparticles may further include a silicon dioxide coating disposed onthe conductive nanoparticle and forming an intermediate shell around theconductive nanoparticle between the conductive nanoparticle and theouter shell, to enable formation of the MIP coating.

(C9) In the method denoted as (C8), the conductive nanoparticle may be ametal nanoparticle.

(C10) In the method denoted as (C9), the metal nanoparticle may includegold.

(C11) In any of the methods denoted as (C7) through (C10), the polymermay be a dielectric polymer.

(C12) In the method denoted as (C11), in the step of exposing, thedielectric polymer may be capable of undergoing hydrogen bonding withthe cotinine.

(C13) In either or both of the methods denoted as (C11) and (C12), thedielectric polymer may include a copolymer.

(C14) In the method denoted as (C13), the copolymer may bepolyvinylpyrrolidone-co-poly(methyl methacrylate).

(C15) In either or both of the methods denoted as (C13) and (C14), thecopolymer may be capable of hydrogen bonding with the cotinine.

(C16) In any of the methods denoted as (C1) through (C15), the MIPcoated conductive nanoparticles may be include any one of the MIP coatedconductive nanoparticles denoted as (A1) through (A8).

(D1) A method for manufacturing a device for detecting cotinine mayinclude (a) making a solution including a polymer, a solvent, andcotinine, (b) adding silicon dioxide coated conductive nanoparticles tothe solution to form a shell, including the polymer and the cotinine,around each of the silicon dioxide coated metal nanoparticles throughbinding of the polymer to the silicon dioxide, so as to formpolymer-silicon dioxide coated conductive nanoparticles, (c) extractingthe polymer-silicon dioxide coated conductive nanoparticles from thesolution, and (d) removing the cotinine from the polymer-silicon dioxidecoated conductive nanoparticles to form molecularly-imprinted-polymer(MIP) coated conductive nanoparticles having (i) specific affinity forbinding with cotinine and (ii) electrical conductivity sensitive to saidbinding with cotinine.

(D2) The method denoted as (D1) may further include depositing the MIPcoated conductive nanoparticles on a substrate to form a film includingthe MIP coated conductive nanoparticles.

(D3) The method denoted as (D3) may further include, before the step ofdepositing, forming two interdigitated electrodes on the substrate, and,in the step of depositing, depositing the MIP coated conductivenanoparticles on the substrate over the interdigitated electrodes suchthat the interdigitated electrodes are located between the substrate andthe film.

(D4) In the method denoted as (D3), the step of depositing may includedrop casting the MIP coated conductive nanoparticles on the substrate.

(D5) In either or both of the methods denoted as (D3) and (D4), the stepof depositing may include dispersing the MIP coated conductivenanoparticles in a solvent to form a MIP coated conductive nanoparticlesolution, and depositing the MIP coated conductive nanoparticle solutionon the substrate.

(D6) Any of the methods denoted as (D1) through (D5) may furtherinclude, before the step of adding, mixing a cross linking agent intothe solution to aid binding of the polymer to the silicon dioxide in thestep of adding.

(D7) In the method denoted as (D6), in the step of mixing, the crosslinking agent may be tetraethyl orthosilicate.

(D8) In any of the methods denoted as (D1) through (D7), the step ofremoving comprising washing the polymer-silicon dioxide coatedconductive nanoparticles with an aromatic solvent.

(D9) In the method denoted as (D8), the aromatic solvent may be selectedfrom the group consisting of toluene and xylene.

(D10) In any of the methods denoted as (D1) through (D9), in the step ofmaking, the polymer may be a dielectric polymer.

(D11) In the method denoted as (D10), the polymer may include acopolymer.

(D12) In the method denoted as (D11), the copolymer may bepolyvinylpyrrolidone-co-poly(methyl methacrylate).

(D13) The MIP coated conductive nanoparticle of claim 52, the copolymerbeing capable of hydrogen bonding with the cotinine.

(D14) In any of the methods denoted as (D1) through (D13), in the stepof making, the solvent may include dichloromethane anddimethylmethacrylate.

(D15) In any of the methods denoted as (D1) through (D14), in the stepof adding, each of the silicon dioxide coated conductive nanoparticlesmay include a metal nanoparticle.

(D16) In the method denoted as (D15), the metal nanoparticle may be agold nanoparticle.

Changes may be made in the above compositions, devices, and methodswithout departing from the scope hereof. It should thus be noted thatthe matter contained in the above description and shown in theaccompanying drawings should be interpreted as illustrative and not in alimiting sense. The following claims are intended to cover generic andspecific features described herein, as well as all statements of thescope of the present compositions, devices, and methods, which, as amatter of language, might be said to fall there between.

1. A molecularly-imprinted-polymer (MIP) coated conductive nanoparticlefor detecting cotinine, comprising: a conductive nanoparticle capable ofconducting an electrical current; a silicon dioxide coating formed onthe conductive nanoparticle and forming a first shell around theconductive nanoparticle; and an MIP coating formed on the silicondioxide coating and forming the second shell, the MIP coating includinga polymer molecularly imprinted with cotinine to provide specificaffinity for binding of cotinine to the MIP coated conductivenanoparticle such that the cotinine is detectable as a change inelectrical conductivity of the MIP coated conductive nanoparticle. 2.The MIP coated conductive nanoparticle of claim 1, the conductivenanoparticle being a metal nanoparticle.
 3. The MIP coated conductivenanoparticle of claim 2, the metal nanoparticle comprising gold.
 4. TheMIP coated conductive nanoparticle of claim 1, the polymer being adielectric polymer.
 5. The MIP coated conductive nanoparticle of claim4, the dielectric polymer being capable of hydrogen bonding with thecotinine.
 6. The MIP coated conductive nanoparticle of claim 5, thedielectric polymer comprising a copolymer.
 7. The MIP coated conductivenanoparticle of claim 6, the copolymer beingpolyvinylpyrrolidone-co-poly(methyl methacrylate).
 8. The MIP coatedconductive nanoparticle of claim 7, the copolymer being capable ofhydrogen bonding with the cotinine.
 9. A device for detecting cotinine,comprising: a film including a plurality of themolecularly-imprinted-polymer (MIP) coated conductive nanoparticles ofclaim 1; and two electrodes in contact with the film for passingelectrical current through the film to detect said binding as a changein electrical conductivity of the film. 10-18. (canceled)
 19. The deviceof claim 9, the electrodes being interdigitated electrodes.
 20. Thedevice of claim 19, each of the interdigitated electrodes having fingerwidth in range from 10 to 100 microns, spacing between theinterdigitated electrodes being in range from 5 to 50 microns.
 21. Thedevice of claim 20, the film having thickness in range from 100nanometers to 1 micron.
 22. The device of claim 9, the film havingthickness in range from 100 nanometers to 1 micron.
 23. The device ofclaim 9, further comprising a receptacle for receiving a liquid sampleand placing the liquid sample in contact with the film to determinepresence of cotinine in the liquid sample.
 24. The device of claim 23,further comprising an enclosure for containing the film and theinterdigitated electrodes, the receptacle being implemented as anopening formed in the enclosure above the film.
 25. The device of claim24, the receptacle having volume capacity in range between 0.5 and 5milliliters.
 26. The device of claim 9, the film and the interdigitatedelectrodes being cooperatively configured to produce a linearrelationship between (a) resistance measured between the interdigitatedelectrodes and (b) concentration of cotinine in a liquid sample placedin contact with the film.
 27. A method for detecting cotinine in aliquid sample, comprising: exposing a film to the liquid sample, thefilm including molecularly-imprinted-polymer (MIP) coated conductivenanoparticles having specific affinity for binding with cotinine, thefilm having electrical conductivity sensitive to said binding, each MIPcoated conductive nanoparticle including: a conductive nanoparticle forproviding electrical conductivity to the MIP coated conductivenanoparticle, an MIP coating forming an outer shell around theconductive nanoparticle, the MIP coating including a polymer molecularlyimprinted with cotinine to provide specific affinity for binding ofcotinine to the MIP coated conductive nanoparticle, and a silicondioxide coating disposed on the conductive nanoparticle and forming anintermediate shell around the conductive nanoparticle between theconductive nanoparticle and the outer shell, to enable formation of theMIP coating; measuring electrical conductivity of the film; anddetermining presence of cotinine in the liquid sample based upon theelectrical conductivity.
 28. The method of claim 27, the step ofmeasuring comprising passing electrical current through the film betweentwo interdigitated electrodes in contact with the film.
 29. The methodof claim 28, the step of measuring comprising measuring the electricalconductivity in form of resistance of the film between theinterdigitated electrodes.
 30. The method of claim 29, the step ofdetermining further comprising: deriving concentration of cotinine inthe liquid sample from the resistance measured in the step of measuring.31. The method of claim 27, the step of exposing further comprising:receiving the liquid sample.
 32. The method of claim 31, the step ofreceiving comprising receiving a volume of the liquid sample in rangefrom 0.5 to 5 milliliters. 33-57. (canceled)